Influence of amino acid relative position on the oxidative modification of histidine and glycine peptides

The radical oxidation of isomeric peptides containing one reactive amino acid [histidine (H)] and another less reactive amino acid [glycine (G)] in the form of dipeptides (HG and GH) and tripeptides (HGG, GHG, and GGH) was studied by mass spectrometry coupled to liquid chromatography (LC-MS) for detection and LC-MSⁿ for structural characterization. The oxidation products identified were keto, hydroxy, keto-hydroxy, and hydroperoxide derivatives for both di- and tripeptides. Among these, it was found that insertion of oxygen atoms occurred at histidine for HG and HGG, and both histidine and glycine for GH, GHG, and GGH. In addition, oxidation products formed by alkoxyl rearrangement reactions with cleavage of the peptide chain were also identified for GH, GHG, and GGH, corroborating hydrogen abstraction step in G residues. These findings were supported through the identification of radical intermediate species formed and trapped with 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) spin trap. The observation of DMPO adducts bearing two spin trap molecules reinforced the abstraction of two hydrogen atoms from the same molecule.     

Reactivity of Tyr–Leu and Leu–Tyr dipeptides: identification of oxidation products by liquid chromatography–tandem mass spectrometry

The exposure of peptides and proteins to reactive hydroxyl radicals results in covalent modifications of amino acid side‐chains and protein backbone. In this study we have investigated the oxidation the isomeric peptides tyrosine–leucine (YL) and leucine–tyrosine (LY), by the hydroxyl radical formed under Fenton reaction (Fe²⁺/H₂O₂). Through mass spectrometry (MS), high‐performance liquid chromatography (HPLC‐MS) and electrospray tandem mass spectrometry (HPLC‐MSⁿ) measurements, we have identified and characterized the oxidation products of these two dipeptides. This approach allowed observing and identifying a wide variety of oxidation products, including isomeric forms of the oxidized dipeptides. We detected oxidation products with 1, 2, 3 and 4 oxygen atoms for both peptides; however, oxidation products with 5 oxygen atoms were only present in LY. LY dipeptide oxidation leads to more isomers with 1 and 2 oxygen atoms than YL (3 vs 5 and 4 vs 5, respectively). Formation of the peroxy group occurred preferentially in the C‐terminal residue. We have also detected oxidation products with double bonds or keto groups, dimers (YL–YL and LY–LY) and other products as a result of cross‐linking. Both amino acids in the dipeptides were oxidized although the peptides showed different oxidation products. Also, amino acid residues have shown different oxidation products depending on the relative position on the dipeptide. Results suggest that amino acids in the C‐terminal position are more prone to oxidation. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of linoleic acid free radicals and other breakdown products using spin trapping with liquid chromatography-electrospray tandem mass spectrometry

Linoleic acid radical products formed by radical reaction (Fenton conditions) were trapped using 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) and analysed by reversed-phase liquid chromatography coupled to electrospray mass spectrometry (LC-MS). The linoleic acid radical species detected as DMPO spin adducts comprised oxidized linoleic acid and short-chain radical species that resulted from the breakdown of carbon and oxygen centred radicals. Based on the m/z values, the short-chain products were identified as alkyl and carboxylic acid DMPO radical adducts that exhibited different elution times. The ions identified as DMPO radical adducts were studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The LC-MS/MS spectra of linoleic acid DMPO radical adducts exhibited the fragment ion at m/z 114 and/or the loss of neutral molecule of 113 Da (DMPO) or 131 Da (DMPO + H2O), indicated to be DMPO adducts. The short-chain products identified allowed inference of the radical oxidation along the linoleic acid chain by abstraction of hydrogen atoms in carbon atoms ranging from C-8 to C-14. Other ions containing the fragment ion at m/z 114 in the LC-MS/MS spectra were attributed to DMPO adducts of unsaturated aldehydes, hydroxy-aldehydes and oxocarboxylic acids. The identification of aldehydic products formed by radical oxidation of linoleic acid peroxidation products, as short-chain product DMPO adducts, is a means of identifying lipid peroxidation products.     

Identification of isomeric spin adducts of Leu-Tyr and Tyr-Leu free radicals using liquid chromatography-tandem mass spectrometry

Free radical species are generally short-lived due to their high reactivity and thus direct measurement and identification are often impossible. In this study we used a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), to trap radical intermediates formed during the oxidation of isomeric dipeptides tyrosine-leucine (Tyr-Leu) and leucine-tyrosine (Leu-Tyr), induced by the hydroxyl radical. To investigate the influence of the amino acid position in the peptide chain on the oxidation and free radical generation, the spin adducts were characterized using LC-MS and MS(n) . We detected carbon and oxygen DMPO adducts and adducts bearing two DMPO, which were analyzed by MS(n) . Both alkoxyl and peroxyl radicals were identified. Radical intermediates were localized in Tyr during oxidation of Tyr-Leu, while radicals were identified in Leu and Tyr during oxidation of Leu-Tyr. DMPO adducts of acyl radical species formed from cleavage of the peptide backbone, promoted by the alkoxyl radical in α carbon of the N-terminal amino acid were observed. The results show that the amino acid position has an influence in the oxidation process, at least on small peptides, and that the α carbon of the N-terminal amino acid is more vulnerable to the attack of the electrophilic hydroxyl radical.     

Liquid chromatography/tandem mass spectrometry analysis of long-chain oxidation products of cardiolipin induced by the hydroxyl radical

The anionic phospholipid cardiolipin (CL) is found almost exclusively in the inner membrane of mitochondria, playing an important role in energy metabolism. Oxidation of CL has been associated with apoptotic events and various pathologies. In this study, electrospray ionization mass spectrometry coupled with liquid chromatography (LC/ESI-MS) was used to identify tetralinoleoyl-cardiolipin (TLCL) modifications induced by the OH(·) radical generated under Fenton reaction conditions (H(2)O(2) and Fe(2+)). The identified oxidation products of TLCL contained 2, 4, 6 and 8 additional oxygen atoms. These long-chain oxidation products were characterized by LC/ESI-MS/MS as doubly [M-2H](2-) and singly charged [M-H](-) ions. A detailed analysis of the fragmentation pathways of these precursor ions allowed the identification of hydroperoxy derivatives of CL. MS/MS analysis indicated that CL oxidation products with 4, 6 and 8 oxygen atoms have one fatty acyl chain bearing 4 oxygen atoms ([RCOO+4O](-)). Even when the TLCL molecule was oxidized by the addition of eight oxygen atoms, one of the acyl chains remained non-modified and one fatty acyl chain contained three or four oxygen atoms. This led us to conclude that under oxidative conditions by the OH(·) radical, the distribution of oxygens/peroxy groups in the CL molecule is not random, even when CL has the same fatty acyl chains in all the positions. Using mass spectrometry, the oxidation products have been unequivocally assigned, which may be useful for their detection in biological samples.     

Ion/molecule reactions of cation radicals formed from protonated polypeptides via gas-phase ion/ion electron transfer

Cation radicals formed via gas-phase electron transfer to multiply protonated polypeptides have been found to react with molecular oxygen. Such cation radicals are of interest within the context of electron transfer dissociation, a phenomenon with high utility for the characterization of peptide and protein primary structures. Most of the cation radicals show the attachment of O(2) under room temperature storage conditions in an electrodynamic ion trap. At higher temperatures and under conditions of collisional activation, the oxygen adduct species lose O(2), HO(*), or HO(2)(*), depending upon the identity of the side chain at the radical site. The fragments containing the C-terminus, the so-called z-ions, which are predominantly radical species, engage in reactions with molecular oxygen. This allows for the facile distinction between z-ions and their complementary even-electron c-ion counterparts. Such a capability has utility in protein identification and characterization via mass spectrometry. Intact electron transfer products also show oxygen attachment. Subsequent activation of such adducts show dissociation behavior very similar to that noted for z-ion adducts. These observations indicate that ion/radical reactions can be used to probe the locations of radical sites in the undissociated electron transfer products as well as distinguish between c- and z-type ions.     

Electrospray mass and tandem mass spectrometry identification of ozone oxidation products of amino acids and small peptides

Aqueous ozonation of the 22 most common amino acids and some small peptides were studied by electrospray mass (ESI-MS) and tandem mass spectrometry. After 5 min of ozonation only His, Met, Trp, and Tyr form oxidation products clearly detectable by ESI-MS. For His, the main oxidation product is formed by the addition of three oxygen atoms, His + 30; for Met and Tyr by the addition of one oxygen atom, Met + O and Tyr + O, and for Trp by the addition of two oxygen atoms, Trp + 20. Ozone oxidation occurs rapidly, products are already detected after 30 s of ozonation, and the reactivity order is Met > Trp > Tyr > His. The structures of the oxygen addition products were investigated by electrospray product ion mass spectra, and by comparing these spectra to those of protonated intact amino acids, and when available, to those of model compounds. His + 30 was assigned as 2-amino-4-oxo-4-(3-formylureido)butanoic acid (1) formed by oxidation of the His imidazole ring, Met + O as methionine sulfoxide (2), Trp + 20 as N-formylkynurenine (4), and Tyr + O as a mixture of dihydroxyphenylalanines (7 and 8). Ozonation of peptides show that the same number of oxygen atoms are added as expected from the ozonation of the free amino acids. The product ion mass spectra of both the protonated intact peptides, MH+, and the main ozonation products (M + nO)H+ (n = 1-3) revealed b and y type ions as the main fragments, which allow one to assign the type and location of modified amino acid in the model peptides.     

Identification of 1‐palmitoyl‐2‐linoleoyl‐phosphatidylethanolamine modifications under oxidative stress conditions by LC‐MS/MS

Phosphatidylethanolamines are a major class of phospholipids found in cellular membranes. Identification of the alterations in these phospholipids, induced by free radicals, could provide new tools for in vivo diagnosis of oxidative stress. In this study, 1‐palmitoyl‐2‐linoleoyl‐phosphatidylethanolamine oxidation products, induced by the hydroxyl radical, were studied using LC‐MS and LC‐MS/MS. Data obtained allowed the identification and separation of isomeric oxidative products with modifications in the sn‐2 acyl chain, attributed to long‐ and short‐chain products. Among long‐chain products keto, keto‐hydroxy, hydroxy, poly‐hydroxy, peroxy and hydroxy–peroxy derivatives were identified. Product ions formed by loss of two H₂O molecules vs loss of HOOH, allowed the identification of, respectively, di‐ (or poli‐) hydroxy vs peroxy derivatives. Location of functional groups was determined by the product ions formed by cleavage of C–C bonds, in the vicinity of the oxidation positions, allowing the identification of C9, C12 and C13 as the predominant substituted positions. Short‐chain products identified comprised aldehydes, hydroxy‐aldehydes and carboxylic derivatives, with modified sn‐2 acyl lengths of C7–C9 and C11, C12. Among the short‐chain products identified, C9 products showed higher relative abundance. Copyright © 2009 John Wiley & Sons, Ltd.     

Tandem mass spectrometry of intact oxidation products of diacylphosphatidylcholines: evidence for the occurrence of the oxidation of the phosphocholine head and differentiation of isomers

Three glycerophosphatidylcholine (GPC) phospholipids (oleoyl-, linoleoyl- and arachidonoylpalmitoylphosphatidylcholine) were oxidized under Fenton reaction conditions (H(2)O(2) and Fe(2+)), and the long-chain oxidation products were detected by electrospray mass spectrometry (ES-MS) and characterized by ES-MS/MS. The intact oxidation products resulted from the insertion of oxygen atoms into the phospholipid structure. The tandem mass spectra of the [MNa](+) molecular ion showed, apart from the characteristic fragments of GPC, fragment ions resulting from neutral losses from [MNa](+), and combined with loss of 59 and 183 Da from [MNa](+). These ions resulted from cleavage of the bond near the hydroxy group by a charge-remote fragmentation mechanism, allowing its location to be pinpointed. The fragments thus formed reflected the positions of the double bonds and of the derivatives along the unsaturated fatty acid chain, giving very useful information, as they allowed the presence of structural isomers and positional isomers to be established. The identification of the fragment ion at m/z 163, which is 16 Da higher than the five-membered cyclophosphane ion (m/z 147), in some tandem mass spectra, is consistent with the oxidation of the phosphocholine head. Some ions were found to occur with the same m/z value; in two of the phospholipids and based on the MS/MS data, structural and positional isomers were differentiated. Our findings indicate that MS/MS is a valuable tool for the identification of the wide complexity of structural features occurring in oxidized phosphatidylcholines during lipid peroxidation in cellular membranes.     

Study of the reaction products of flavonols with 2,2-diphenyl-1-picrylhydrazyl using liquid chromatography coupled with negative electrospray ionization tandem mass spectrometry

The products obtained after the reaction between flavonols and the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH(*)) in both methanol and acetonitrile were characterized using liquid chromatography coupled with negative electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) and NMR spectroscopy. The flavonols studied were quercetin, kaempferol and myricetin. In methanol, two reaction products of oxidized quercetin were identified using LC/ESI-MS/MS and NMR. Quercetin was oxidized through a transfer of two H-atoms to DPPH(*) and subsequently incorporated either two CH(3)OH molecules or one CH(3)OH- and one H(2)O molecule giving the products 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-2,3-dimethoxy-2,3-dihydrochromen-4-one and 2-(3,4-dihydroxyphenyl)-3,3,5,7-tetrahydroxy-2-methoxy-2,3-dihydrochromen-4-one, respectively. LC/ESI-MS/MS analysis revealed that in methanol, kaempferol and myricetin also gave rise to methoxylated oxidation products similar to that identified for quercetin. Kaempferol, in addition, also exhibited products where a kaempferol radical, obtained by a transfer of one H-atom to DPPH(*), reacted with CH(3)OH through the addition of CH(3)O(*), yielding two isomeric products. When the reaction took place in acetonitrile, LC/ESI-MS/MS analysis showed that both quercetin and myricetin formed stable isomeric quinone products obtained by a transfer of two H-atoms to DPPH(*). In contrast, kaempferol formed two isomeric products where a kaempferol radical reacted with H(2)O through the addition of OH(*), i.e. similar to the reaction of kaempferol radicals with CH(3)OH.     

Interstitial OH radicals in F2-laser-irradiated bulk amorphous SiO2

An interstitial hydroxyl radical (HO*) has been generated in bulk amorphous SiO2 (a-SiO2) loaded with interstitial H2O molecules and exposed to F2 laser light (hnu = 7.9 eV, lambda = 157 nm) at 77 K. F2 laser light dissociates an O-H bond of interstitial H2O into a pair of hydrogen atom (H0) and HO*. The resultant H0 disappears below 150 K, whereas HO* is detectable after thermal annealing at 200 K. The electron paramagnetic resonance (EPR) signal of the interstitial HO* recorded at 77 K is similar to that formed in amorphous ice, indicating that HO* is confined in an orthorhombic field by hydrogen bonding, probably with adjacent H2O molecules, silanol (SiOH) groups, and bridging oxygen atoms in the a-SiO2 network.     

Hydrogen isotope effects and mechanism of aqueous ozone and peroxone decompositions

Hydrogen peroxide exalts the reactivity of aqueous ozone by reasons that remain obscure. Should H2O2 enhance free radical production, as it is generally believed, a chain mechanism propagated by (.OH/.O2-) species would account for O3 decomposition rates in neat H2O, HR-O3, and in peroxone (O3 + H2O2) solutions, HPR-O3. We found, however, that: (1) the radical mechanism correctly predicts HR-O3 but vastly overestimates HPR-O3, (2) solvent deuteration experiments preclude radical products from the (O3 + HO2-) reaction. The modest kinetic isotope effect (KIE) we measure in H2O/D2O: HR-O3/DR-O3 = 1.5 +/- 0.3, is compatible with a chain process driven by electron- and/or O-atom transfer processes. But the large KIE found in peroxone: HPR-O3/DPR-O3 = 19.6 +/- 4.0, is due to an elementary (O3 + HO2-) reaction involving H-O2- bond cleavage. Since the KIE for the hypothetical H-atom transfer: O3 + HO2- HO3. +.O2-, would emerge as a KIE1/2 factor in the rates of the ensuing radical chain, the magnitude of the observed KIE must be associated with the hydride transfer reaction that yields a diamagnetic species: O3 + HO2- HO3- + O2. HO3-/H2O3 may be the bactericidal trioxide recently identified in the antibody-catalyzed addition of O2(1Deltag) to H2O.     

Reaction mechanisms of a cyclic ether intermediate: Ethyloxirane

Oxiranes are a class of cyclic ethers formed in abundance during low‐temperature combustion of hydrocarbons and biofuels, either via chain‐propagating steps that occur from unimolecular decomposition of β‐hydroperoxyalkyl radicals (β‐?QOOH) or from reactions of HO? with alkenes. Ethyloxirane is one of four alkyl‐substituted cyclic ether isomers produced as an intermediate from n‐butane oxidation. While rate coefficients for β‐?QOOH → ethyloxirane + ?H are reported extensively, subsequent reaction mechanisms of the cyclic ether are not. As a result, chemical kinetics mechanisms commonly adopt simplified chemistry to describe ethyloxirane consumption by convoluting several elementary reactions into a single step, which may introduce mechanism truncation error—uncertainty derived from missing or incomplete chemistry. The present work provides fundamental insight on reaction mechanisms of ethyloxirane in support of ongoing efforts to minimize mechanism truncation error. Reaction mechanisms are inferred from the detection of products during chlorine atom‐initiated oxidation experiments using multiplexed photoionization mass spectrometry conducted at 10 Torr and temperatures of 650 K and 800 K. To complement the experiments, calculations of stationary point energies were conducted using the ccCA‐PS3 composite method on ?R + O₂ potential energy surfaces for the four ethyloxiranyl radical isomers, which produced barrier heights for 24 reaction pathways. In addition to products from ?QOOH → cyclic ether + ?H and ?R + O₂ → conjugate alkene + HO?, both of which were significant pathways and are prototypical to alkane oxidation, other species were identified from ring‐opening of both ethyloxiranyl and ?QOOH radicals. The latter occurs when the unpaired electron is localized on the ether group, causing the initial ?QOOH structure to ring‐open and form a resonance‐stabilized ketohydroperoxide‐type radical. The present work provides the first analysis of ethyloxirane oxidation chemistry, which reveals that consumption pathways are complex and may require an expansion of submechanisms to increase the fidelity of chemical kinetics mechanisms.     

Uncovering the fundamental chemistry of alkyl + O2 reactions via measurements of product formation

The reactions of alkyl radicals (R) with molecular oxygen (O(2)) are critical components in chemical models of tropospheric chemistry, hydrocarbon flames, and autoignition phenomena. The fundamental kinetics of the R + O(2) reactions is governed by a rich interplay of elementary physical chemistry processes. At low temperatures and moderate pressures, the reactions form stabilized alkylperoxy radicals (RO(2)), which are key chain carriers in the atmospheric oxidation of hydrocarbons. At higher temperatures, thermal dissociation of the alkylperoxy radicals becomes more rapid and the formation of hydroperoxyl radicals (HO(2)) and the conjugate alkenes begins to dominate the reaction. Internal isomerization of the RO(2) radicals to produce hydroperoxyalkyl radicals, often denoted by QOOH, leads to the production of OH and cyclic ether products. More crucially for combustion chemistry, reactions of the ephemeral QOOH species are also thought to be the key to chain branching in autoignition chemistry. Over the past decade, the understanding of these important reactions has changed greatly. A recognition, arising from classical kinetics experiments but firmly established by recent high-level theoretical studies, that HO(2) elimination occurs directly from an alkylperoxy radical without intervening isomerization has helped resolve tenacious controversies regarding HO(2) formation in these reactions. Second, the importance of including formally direct chemical activation pathways, especially for the formation of products but also for the formation of the QOOH species, in kinetic modeling of R + O(2) chemistry has been demonstrated. In addition, it appears that the crucial rate coefficient for the isomerization of RO(2) radicals to QOOH may be significantly larger than previously thought. These reinterpretations of this class of reactions have been supported by comparison of detailed theoretical calculations to new experimental results that monitor the formation of products of hydrocarbon radical oxidation following a pulsed-photolytic initiation. In this article, these recent experiments are discussed and their contributions to improving general models of alkyl + O(2) reactions are highlighted. Finally, several prospects are discussed for extending the experimental investigations to the pivotal questions of QOOH radical chemistry.     

Tandem mass spectrometry of kahalalides: identification of two new cyclic depsipeptides, kahalalide R and S from Elysia grandifolia

Spectra obtained using electrospray ionization mass spectrometry (ESI-MS) of the mollusk Elysia grandifolia showed a cluster of molecular ion peaks centered at a molecular mass of 1478 Da (kahalalide F, an anticancer agent). Two new molecules, kahalalide R (m/z 1464) and S (m/z 1492) were characterized using tandem mass spectrometry. The mass differences of 14 Da suggest that they are homologous molecules. In addition, previously identified kahalalide D and kahalalide G are also reported. However, the ESI-MS of the mollusk's algal diet Bryopsis plumosa showed the presence of only kahalalide F. The amino acid sequences of kahalalide R and S are proposed using collision-induced dissociation (CID) experiments of singly and doubly charged molecular ions and by comparison with the amino acid sequence of kahalalide F. The pathway is presented for the loss of amino acid residues in kahalalide F. It is observed that there is sequential loss of amino acids in the linear peptide chain, but in the cyclic part the ring opens at the amide bond rather than at the lactone linkage, and the loss of amino acid residues is not sequential. The CID experiment of the alkali-metal-cationized molecular ions shows that the sodium and potassium ions coordinate to the amide nitrogen/oxygen in the linear peptide chain of the molecule and not to the lactone oxygen of the lactone. In the case of kahalalide D, CID of the protonated peptide opens the depsipeptide ring to form a linear peptide with acylium ion, and fragment ion signals indicate losses of amino acids in sequential order. In this study, tandem mass spectrometry has provided the detailed information required to fully characterize the new peptides.     

Separation of peroxidation products of diacyl-phosphatidylcholines by reversed-phase liquid chromatography-mass spectrometry

Lipid peroxidation process has attracted much attention due to the growing evidence of its involvement in the pathogenesis of age-related diseases. The monitoring of the lipid peroxidation products in phospholipids, formed under oxidative stress conditions, may provide new markers for oxidative stress signaling and for disease states, giving new insights in the pathogenesis process. Reversed-phase liquid chromatographic method coupled to mass spectrometry was developed for the separation of oxidized glycero-phosphatidylcholine (GPC) peroxidation products formed by the Fenton reaction that mimic in vivo oxidative stress conditions. The LC-MS conditions were applied for the separation of peroxidation products of oleoyl- (POPC), lineloyl- (PLPC) and arachidonoyl-palmitoyl phosphatidylcholine (PAPC). The peroxidation products separated included products resulting from the insertion of oxygen atoms in the sn-2 chain (long-chain), and products with the sn-2 chain shortened resulting from cleavage of oxygen-centered radicals (short-chain). Among long-chain products were the keto, hydroxy, hydroperoxide and poly-hydroxy derivatives, while short-chain products included dicarboxylic acids, aldehydes and hydroxy-aldehydes. Separation of long-chain products formed in each phosphatidylcholine was observed, and the reconstructed ion chromatogram of each ion showed an increase in the number of peaks with the increase in the number of oxygen atoms inserted into the phospholipid. Separation of short-chain products took place according to the functional group present at the sn-2 moiety that allowed the elution of dicarboxylic acids distinct from aldehydes. Separation between isomeric structures that were present in short- and long-chain products was also achieved.     

Edman降解中异硫氰酸苯酯新修饰位点的发现和验证

Edman降解是最早建立的一种用于多肽和蛋白质氨基端测序的方法,该方法现在仍被广泛用于生物化学领域。随着高通量蛋白质组学技术的发展和应用,该方法中的异硫氰酸苯酯反应被用于修饰蛋白质氨基端,并用于检测蛋白质水解位点。但还没有异硫氰酸苯酯是否可以修饰其他氨基酸侧链并影响多肽序列分析的研究。为了探究其修饰其他氨基酸的可能性,本文利用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）和液相色谱-串联质谱（LC-MS/MS）研究了异硫氰酸苯酯对一个模型肽的化学修饰。质谱数据解析后发现在高浓度异硫氰酸苯酯的反应条件下,组氨酸上可以引入一个新的异硫氰酸苯酯修饰位点。这一修饰位点的发现预示着通过改变实验条件或分析方法,可以更准确地利用Edman降解和蛋白质组学技术分析多肽和蛋白质。     

OH radical oxidation of the sorbitylfurfural furanic ring to sugar derivatives induced by radiolysis in aerobic environment

Chemical radiolytic oxidation induced by OH addition on 1-(2-furan-2-yl-5-hydroxy-6-hydroxymethyl-[1,3]dioxan-4-yl)-ethan-1,2-diol (sorbitylfurfural, SF) leads, in the presence of controlled amounts of oxygen, to a permanent functional modification of the target molecule. The yield of conversion reaches 60% of the starting material. LC-MS analysis allowed the identification, as final products, of carboxylic acids, butenal and hydroxy-furan derivatives in which the sugar chain remains unbroken, while the furanic ring is attacked first by OH and then by oxygen, giving in succession an intra-/inter-molecular rearrangement of the allylperoxyl radicals thus formed. The proposed oxidation of the furanic ring envisages the peroxyl intermediates undergoing mono- and/or bi-molecular reactions; a reaction path has been outlined and is reported here. The presence of unsaturated bonds in the final products could provide a further site for radical scavenger activity. Therefore, the fast reaction with O₂ and the rearrangement of the produced peroxyl radicals to species, which are likely to be effective OH-capturers, reinforces the antioxidant ability of SF.     

Coordination of copper(II) ions by the fragments of neuropeptide gamma containing D1, H9, H12 residues and products of copper-catalyzed oxidation

A potentiometric, spectroscopic (UV-Vis, CD and EPR) and mass spectrometric (ESI-MS) study of Cu(II) binding to the (1-2,7-21)NPG, Asp(1)-Ala-Ile(7)-Ser-His(9)-Lys-Arg-His(12)-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met(21)-NH(2), and Ac-(1-2,7-21)NPG, Ac-Asp(1)-Ala-Ile(7)-Ser-His(9)-Lys-Arg-His(12)-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met(21)-NH(2), fragments of neuropeptide gamma were carried out. The results clearly indicate the stabilization of the 1 N {NH(2), β-COO(-)}, 2 N {NH(2), β-COO(-), N(Im)} and 3 N {NH(2), β-COO(-), 2N(Im)} complexes by the coordination of the β-carboxylate group of the D(1) residue. For the (1-2,7-21)NPG the CuH(2)L complex with 3 N {NH(2), β-COO(-), 2N(Im)}, the binding mode dominates in a wide pH range of 4-8.5. With the sequential increase of pH, deprotonated amide nitrogens are involved in copper coordination. For the Ac-(1-2,7-21)NPG peptide the imidazole nitrogen atoms are the primary metal binding sites forming macrochelates in the pH range 4 to 7. The CuHL complex with 4 N {N(Im), N(-), N(-), N(Im)} coordination mode is formed in pH range 6-9. Deprotonation and co-ordination of the third amide nitrogen were detected at pH ～8.6. Metal-catalyzed oxidation (MCO) of proteins is mainly a site-specific process in which one or a few amino acids at metal-binding sites on the protein are preferentially oxidized. To elucidate the products of the copper(II)-catalyzed oxidation of the (1-2,7-21)NPG and Ac-(1-2,7-21)NPG, the liquid chromatography-mass spectrometry (LC-MS) method and Cu(II)/hydrogen peroxide as a model oxidizing system were employed. In the presence of hydrogen peroxide with 1?:?4 peptide-H(2)O(2) molar ratio for the Ac-(1-2,7-21)NPG peptide the oxidation of the methionine residue to methionine sulfoxide and for (1-2,7-21)NPG to sulfone was observed. For the Cu(II)-peptide-hydrogen peroxide in 1?:?1?:?4 molar ratio systems, oxidation of the histidine residues to 2-oxohistidines was detected. Under experimental conditions the (1-2,7-21)NPG and Ac-(1-2,7-21)NPG undergo fragmentations by cleavage of the S(8)-H(9), H(9)-K(10), R(11)-H(12) and H(12)-K(13) peptide bonds supporting the participation of the H(9) and H(12) residues in the coordination of copper(II) ions. For the (1-2,7-21)NPG peptide chain the involvement of the D(1) residue in the coordination of metal ions is supported by the alkoxyl radical modification of this amino acid residue.     

Partial oxidation of propene on oxygen-covered Au(111)

Partial oxidation of propene is promoted by Au following deposition of atomic oxygen (0.3 ML) via O3 decomposition on Au(111) at 200 K. Several partial oxidation products--acrolein, acrylic acid, and carbon suboxide (O=C=C=C=O)-are produced in competition with combustion to CO2 and H2O. Acrolein is the primary partial oxidation product, and it is further oxidized to the other products by excess oxygen. We propose that acrolein is derived from allyloxy intermediate that is formed via insertion of oxygen into the allylic C-H bond. While no propene epoxide formation is detected from oxidation of C3H6, a small amount of epoxidation is observed during reaction of C3D6 and CD3CH=CH2. These results are strong indications that small changes in the energy required for allylic C-H activation, in this case due to a kinetic isotope effect, may dramatically change the selectivity; thus, small modifications of the properties of oxygen on Au may lead to the more desirable epoxidation process. Our results are discussed in the context of the origin of activity of Au-based catalysts.